The clinical significance and oncogenic function of LRRFIP1 in pancreatic cancer

Purpose Pancreatic cancer is a lethal malignancy with a grim prognosis. Previous studies have proven that Leucine Rich Repeat of Flightless-1 Interacting Protein 1 (LRRFIP1) plays a pivotal role in cell biological processes, while its clinical significance and function in pancreatic cancer remain to be elucidated. Hence, we aimed to explore the roles and mechanisms of LRRFIP1 in pancreatic cancer. Methods The expression of LRRFIP1 in pancreatic cancer tissues and its clinical significance for pancreatic cancer were analyzed by immunohistochemistry assay and bioinformatic analysis. The influences of LRRFIP1 on the proliferation and migration of pancreatic cancer cells were assessed in vitro. The underlying mechanisms of LRRFIP1 in pancreatic cancer progression were explored using gene set enrichment analysis (GSEA) and molecular experiments. Results The results showed that LRRFIP1 expression was significantly upregulated in pancreatic cancer tissues compared to the normal tissues, and such upregulation was associated with poor prognosis of patients with pancreatic cancer. GSEA revealed that LRRFIP1 upregulation was significantly associated with various cancer-associated signaling pathways, including PI3K/AKT signaling pathway and Wnt pathway. Furthermore, LRRFIP1 was found to be associated with the infiltration of various immune cells. Functionally, LRRFIP1 silencing suppressed cell proliferation somewhat and inhibited migration substantially. Further molecular experiments indicated that LRRFIP1 silencing inactivated the AKT/GSK-3β/β-catenin signaling axis. Conclusion Taken together, LRRFIP1 is associated with tumorigenesis, immune cell infiltration, and prognosis in pancreatic cancer, which suggests that LRRFIP1 may be a potential biomarker and therapeutic target for pancreatic cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-00977-3.


Introduction
Pancreatic cancer is one of the most lethal cancers in the world, with a 5-year survival rate of approximately 10% [1].Surgical intervention is a top priority in the treatment of pancreatic cancer, but most of the patients are diagnosed at an advanced stage, missing out on surgery.In recent years, immunotherapy, such as immune checkpoint blockade, has achieved remarkable efficacy in many malignancies [2], but it remains a major challenge due to the lack of specific biomarkers for pancreatic cancer.Currently, carcinoembryonic antigen 19-9 (CA19-9) is the most commonly used tumor-associated antigen for the serological diagnosis and prognosis evaluation of pancreatic cancer [3], but its specificity in distinguishing pancreatic cancer from biliary diseases is not satisfactory.Therefore, it is imperative to identify some novel biomarkers and therapeutic targets for improving the diagnosis and prognosis of pancreatic cancer.
The Leucine Rich Repeat of Flightless-1 Interacting Protein 1/GC-binding factor 2 (LRRFIP1/GCF2) gene contains eight exons and introns and encodes 752 amino acid residues with a detected molecular mass of 160 kDa.The molecular structure of the LRRFIP1 protein consists of three domains: a helix domain, a central coiled-coil domain, and a nucleic acid binding domain, which is correlated with its functions such as transcriptional inhibition, signal transduction, and cytoskeleton rearrangement [4].Some studies have revealed that LRRFIP1 is an oncogene that is highly expressed in multiple malignant tumors [4][5][6][7].Moreover, some researchers have confirmed that high LRRFIP1 expression is associated the cancer progression, including cancer cell proliferation, invasion, metastasis, and drug resistance [7][8][9][10][11].In pancreatic cancer cells, silencing of LRRFIP1 can reverse the epithelial-mesenchymal transition (EMT) [12], and increase gemcitabine sensitivity [13].Therefore, LRRFIP1 plays a critical role in pancreatic cancer.However, the clinical significance and function of LRRFIP1 in pancreatic cancer are yet to be defined.
In the present study, we evaluated LRRFIP1 expression in pancreatic cancer tissues and investigated its clinical significance.Furthermore, bioinformatics analysis was used to investigate the signaling pathways that LRRFIP1 may be involved in, as well as its possible role in immune cell infiltration.Lastly, in vitro studies were performed to explore its biological functions and underlying mechanisms in pancreatic cancer progression.

TCGA and GEO datasets analyses
Transcriptional expression data of pancreatic cancer and corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA) portal (https:// portal.gdc.cancer.gov/), which contains 179 pancreatic cancer samples and 4 paracancerous tissues.Meanwhile, 167 normal samples were obtained from Genotype-Tissue Expression (GTEx) database (https:// gtexp ortal.org/).For further analysis, RNA-Seq data with a workflow type of Fragments Per Kilobase per Million (FPKM) was transformed into Transcripts per million reads (TPM) format, followed by log 2 conversion [15].Gene Expression Omnibus (GEO) is a public comprehensive gene expression library in the National Center of Biotechnology Information (https:// www.ncbi.nlm.nih.gov/ geo/) [16].The GEO dataset GSE16515 [17] was chosen to analyze LRRFIP1 mRNA expression in pancreatic cancer and normal tissues.

Enrichment analysis
To investigate the potential biological function of LRRFIP1 in pancreatic cancer, we selected the top 210 genes that were most positively correlated with LRRFIP1 in TCGA pancreatic cancer for enrichment analysis.The EnrichGO function of the R package "clusterProfiler" was used to perform gene ontology (GO) enrichment, including biological process (BP), cell component (CC), molecular function (MF), and Kyoto encyclopedia of genes and genomes (KEGG) [18].In addition, the EnrichKEGG function in the R package "clusterProfiler" was used to perform KEGG analysis.

Construction of protein-protein interaction (PPI) networks
STRING (https:// www.string-db.org/) is an online database for analyzing protein-protein interaction networks.In this study, we utilized STRING to search the proteins that interacted with LRRFIP1 (interaction score > 0.70) and construct a PPI network.We also analyzed the correlation between LRRFIP1 and the top ten possible interacting proteins according to the PPI network.

Gene set enrichment analysis (GSEA)
GSEA was performed with the R package "clusterProfiler" to analyze the differences in function and pathway terms between the high-LRRFIP1 and low-LRRFIP1 expression groups.A function or pathway term with a p-value < 0.05 was considered to be a statistically significant enrichment.

Immune cell infiltration analysis
To evaluate the role of LRRFIP1 expression in immune infiltration in pancreatic cancer, we investigated the association between LRRFIP1 expression and 24 different types of immune cells using the single-sample GSEA (ssGSEA) method in the R package GSVA (Version 1.34.0)[19,20].Moreover, the infiltration levels of immune cells in high-and low-LRRFIP1 expression groups were compared.

Cell migration assays
For wound healing assay, cells were seeded into a 6-well plate and transfected with siLRRFIP1 or siNC.When the cells achieved 90% confluence, they were scratched with a 200-μL pipette tip and subsequently cultured in the serum-free medium.After 24 h, images of the wound healing were taken and quantified using ImageJ software.The wound healing rate was calculated as follows: wound healing rate = (initial area of the wound-the wound area at 24 h)/initial area of the wound × 100%.In addition, transwell cell assay was performed in the transwell system with an 8-μm pore membrane (JET BIOFIL, China).Briefly, the transfected cells (1.5 × 10 5 cells/well) were seeded in the upper chamber with serum-free culture medium, while the lower chamber contained medium with 20% fetal bovine serum.At 48 h, the migrated cells were fixed with 4% paraformaldehyde for 15 min, followed by staining with 0.1% crystal violet for 10 min at room temperature, and then images were taken with an Olympus CKX53 inverted microscope.The number of migrated cells was quantified by ImageJ.

Statistical analysis
R (V 3.6.3)was used for all statistical analyses.The differences between two sets of data were compared using Wilcoxon rank sum test or Student's t-test, where appropriate.A value of p < 0.05 was considered as a statistical difference.

LRRFIP1 is highly expressed in pancreatic cancer tissues
To investigate LRRFIP1 mRNA and protein expression in pancreatic cancer, we extracted LRRFIP1 expression data from TCGA, GEO, CPTAC, and HPA.The results showed that compared to normal tissues, LRRFIP1 mRNA expression was significantly upregulated in pancreatic cancer tissues (Fig. 1A, B , p< 0.05).Similar results were obtained on the protein expression level of LRRFIP1 (Fig. 1C, D, and Fig. S1).Given that LRRFIP1 expression was upregulated in pancreatic cancer tissues, we used receiver operating characteristic (ROC) curve analysis to assess LRRFIP1's diagnostic value for pancreatic cancer.The result showed that LRRFIP1 had an area under the curve (AUC) of 0.969 (confidence interval (CI): 0.949-0.990)(Fig. 1E) in discriminating pancreatic cancer tissues from normal tissues.Collectively, these results suggest that the expression of LRRFIP1 is upregulated in pancreatic cancer and LRRFIP1 has the potential to distinguish pancreatic cancer patients from healthy subjects.

The LRRFIP1-correlated genes are enriched in various biological terms in pancreatic cancer
To investigate the potential functions of LRRFIP1 in pancreatic cancer, we searched the TCGA database for LRRFIP1 expression-correlated genes in the pancreatic cancer cohort to analyze the involved pathways.We identified the top 210 genes that most positively correlated with LRRFIP1, and these genes were subjected to gene ontology (GO) and KEGG enrichment analysis using the "clusterProfile" R package.The GO analysis showed that the majority of the genes were associated with multiple molecular functions, including "serine hydrolase activity", "serine-type peptidase activity", and "serine-type endopeptidase activity" (Fig. 3A).Meanwhile, the KEGG data revealed that LRRFIP1 may be related to "pancreatic secretion", "protein digestion and absorption", and "fat digestion and absorption" (Fig. 3B).We further used the STRING database to build a PPI network of LRRFIP1 and the top ten potential interacting proteins were shown in Fig. 4A.The correlation analyses between LRRFIP1 expression and some potential interacting proteins (r ≥ 0.4 and p < 0.001) were shown in Fig. 4B-H.We further verified the effects of LRRFIP1 on the expression of these genes.Consistent with bioinformatics predictions, qRT-PCR assay showed that LRRFIP1-knockdown significantly downregulated mRNA expression of most of the genes in MIAPaCa-2 cells (Fig. S2).Altogether, the above data demonstrate that LRRFIP1 may play an important role in pancreatic cancer by regulating various signaling pathways.and "Interferon alpha/beta signalling" were identified enriched in the pancreatic cancer patients with high LRRFIP1 expression (Fig. 5A-I).

LRRFIP1 predicts immune cell infiltration in pancreatic cancer
Previous studies reported that LRRFIP1 induces the production of type 1 interferon (IFN) and pro-inflammatory cytokines via binding nucleic acids in infectious diseases [22,23].Furthermore, LRRFIP1 has recently been identified as the key response prediction gene of PD-1 inhibitors in glioblastoma [24].Therefore, we analyzed the relationship between LRRFIP1 expression and the immune cells.We applied the R package ssGSEA to investigate the association between the LRRFIP1 expression and 24 different types of immune cells.As shown in Fig. 6A, LRRFIP1 expression was positively correlated with infiltration levels of Th2 cells, T helper cells, Tcm, Th1 cells, macrophages, and neutrophils (p < 0.05 for all), whereas it was negatively correlated with infiltration levels of pDC, TFH, cytotoxic cells, CD8 T cells, Th17 cells, B cells, and T cells (p < 0.05 for all).Further analysis showed that the infiltration level of pDC was significantly lower in the group of high LRRFIP1 expression than that of low LRRFIP1 expression (p < 0.001, Fig. 6B).Similar results were observed in TFH cells (p < 0.01, Fig. 6C), and cytotoxic cells (p < 0.05, Fig. 6D).Conversely, as shown in Fig. 6E-G, the infiltration levels of Th2 cells, Tcm cells, and T helper cells were significantly higher in the group of high LRRFIP1 expression than that of low LRRFIP1 expression (p < 0.05 for all).In short, these results suggest that high LRRFIP1 expression is related to immune cell infiltration in pancreatic cancer.

Knockdown of LRRFIP1 inhibits proliferation and migration in pancreatic cancer cells.
To this end, we set out to investigate the functional role of LRRFIP1 in pancreatic cancer cell models.To evaluate the effects of LRRFIP1 on the growth and metastatic potential of pancreatic cancer cells, we knocked down LRRFIP1 in MIAPaCa-2 and PANC-1 cells (Fig. 7A).The results showed that attenuated LRRFIP1 expression significantly suppressed cell viability (Fig. 7B, p < 0.05 for both) and migration ability (Fig. 7C, D, p < 0.05 for all).Taken together, these data indicate that LRRFIP1 may play an oncogenic role in tumorigenesis and metastatic potential of pancreatic cancer.

LRRFIP1 silencing dampens AKT/GSK-3β/β-catenin signaling in pancreatic cancer cells
Given that the above results showed that AKT and Wnt signaling were enriched in pancreatic cancer patients with high LRRFIP1 expression (Fig. 5B, D), and the roles of AKT and Wnt signalings in cancer progression are well established [25,26].We measured the protein levels of AKT/GSK-3β/β-catenin signaling in LRRFIP1-silenced cells.Western blotting assay revealed that knockdown of LRRFIP1 resulted in a notable downregulation in p-AKT, p-GSK-3β, and β-catenin in MIAPaCa-2 cells (Fig. 8).Taken together, the findings suggest that LRRFIP1 may influence cell proliferation and mobility in pancreatic cancer through regulation of AKT/GSK-3β/β-catenin signaling pathway.

Discussion
In the present study, we revealed that LRRFIP1 was upregulated in pancreatic cancer, and its upregulation was associated with poor overall survival, which indicated that LRRFIP1 could act as a prognosis predictor for pancreatic cancer.Moreover, LRRFIP1 was revealed to be associated with the infiltration of multiple immune cell types.Based on the findings that LRRFIP1 might get involved in tumorigenesis revealed by GSEA analysis, subsequent in vitro experiments were conducted, which demonstrated that LRRFIP1 was involved in the progression of pancreatic cancer.Our findings revealed that LRRFIP1 could promote pancreatic cancer progression through the AKT/GSK-3β/β-catenin signaling axis.LRRFIP1 is abnormally expressed in a variety of cancers [4][5][6][7] and regulates a wide range of biological systems and processes, including immune response to microorganisms and autoimmunity, remodeling of cytoskeletal system, signal transduction pathways, and transcriptional regulations of genes [8][9][10][11].In the case of pancreatic cancer, silencing of LRRFIP1 can reverse the EMT of pancreatic cancer cells and increase the gemcitabine sensitivity in pancreatic cancer cells [12,13].However, its clinical value for pancreatic cancer diagnosis and prognosis has yet to be determined.Here, we found that LRRFIP1 was significantly upregulated in pancreatic cancer and it could distinguish pancreatic cancer tissues from normal tissues according to ROC analysis.Further research should be performed to confirm its clinical significance for pancreatic cancer diagnosis.
In terms of prognosis, upregulation of LRRFIP1 has been associated with poor overall survival in gliomas [27] and hepatocellular carcinoma [28].In this study, Kaplan-Meier survival analysis showed that the pancreatic cancer patients with high LRRFIP1 expression had a shorter overall survival than those patients with low LRRFIP1 expression.Similarly, several clinicopathologic subgroups with high LRRFIP1 expression showed lower overall survival than those with low LRRFIP1 expression.Collectively, the above results suggest that LRRFIP1 is an important prognosis biomarker for pancreatic cancer.
Previous studies have reported that LRRFIP1 acts as a mediator in multiple signaling transduction pathways [4].To explore the functions of LRRFIP1 in pancreatic cancer, we identified the top 210 genes that most positively correlated with LRRFIP1, and these genes were subjected to GO and KEGG enrichment analysis using the "clusterProfile" R package.GO and KEGG enrichment analysis suggests that the LRRFIP1 was involved with "serine hydrolase activity", "serine-type peptidase activity", "serine-type endopeptidase activity", "pancreatic secretion", "protein digestion and absorption", and "fat digestion in pancreatic cancer".The role of LRRFIP1 in these biological terms requires more clarification.We also found ten possible interacting proteins of LRRFIP1 based on PPI networks, including DHX9, DHX36, and MYD88.Furthermore, qRT-PCR demonstrated that knocking down LRRFIP1 decreased the expression of the majority of the interacting molecules.More research is needed to determine how LRRFIP1 affects the expression of these molecules, as well as whether these molecules mediate LRRFIP1's effects on pancreatic cancer.
Immune cell infiltration is one of the hallmarks of solid tumors and plays a critical role in disease progression [29].Because LRRFIP1 has been linked to immune responses in infectious diseases and glioblastoma [22][23][24], we proposed the hypothesis that it may play a role in pancreatic cancer tumor immunity.The results of the correlation analysis revealed that high LRRFIP1 expression was negatively correlated with infiltration levels of pDC, TFH, and Cytotoxic cells, while it was positively related to the infiltration levels of Tcm, T helper cells, and Th2 cells; however, the difference in many immune cell populations between tumors with low and high LRRFIP1 expression is modest.It is worth investigating whether it is functionally relevant to pancreatic cancer's malignant behaviors.It is reported that LRRFIP1 can interact with MYD88 [30], a key regulator of DC and Th2 cells [31], and trigger TLR-mediated NF-κB activity in macrophage and primary human PBMCs [30].However, we found that in tumors with high LRRFIP1 expression, pDC cell infiltration decreased while Th2 cell infiltration increased.We currently do not understand the reasons for this association in pancreatic cancer.Thus, more research is needed to identify whether LRRFIP1 has a regulatory role in the modulation of MyD88 signaling in DC and Th2 cells.
Finally, the results of GSEA identified that nine pathways were associated with LRRFIP1 in pancreatic cancer, including "pancreatic cancer signaling", "Wnt signaling", Hedgehog signaling pathway", "PI3K/AKT/mTOR_VITD3 signaling", "Integrin signaling pathway, "Integrin-mediated cell adhesion signaling", "RhoA pathway", "Toll-like receptor signaling related to MYD88", and "Interferon alpha/beta signaling".The functions of LRRFIP1 in some of these signaling have been proved in recent studies [9][10][11][12][13].Therefore, it is a reasonable prospect that LRRFIP1 plays multiple roles in the biological process of pancreatic cancer.Here, we found that knockdown of LRRFIP1 in pancreatic cancer cells downregulated the protein levels of p-AKT, p-GSK-3β, and β-catenin, which suggests that knockdown of LRRFIP1 inhibited the proliferation and migration via dampening the activation of AKT/GSK-3β/β-catenin signaling in pancreatic cancer cells.In support, LRRFIP1 was reported to promote cell invasion and migration through the PI3K/AKT pathway in vascular smooth muscle cells [32].More research is needed to determine whether LRRFIP1 has a regulatory role in other GSEA-identified pathways.https://doi.org/10.1007/s12672-024-00977-3 It is worth mentioning that LRRFIP1 knockdown had a greater inhibitory effect on cell mobility than on cell proliferation, with cell proliferation being moderately suppressed, indicating that LRRFIP1 plays a dominating role in cell mobility rather than cell proliferation in pancreatic cancer.
We have to point out there are some limitations in this study.The results and implications regarding LRRFIP1 expression and prognostic value in pancreatic cancer were mainly based on an analysis of online public databases and a small cohort of pancreatic cancer patients; therefore, additional research involving larger independent cohorts should be conducted to validate these findings and implications.Moreover, more in vivo and in vitro experiments are required to clarify the role of LRRFIP1 in pancreatic cancer, especially immune infiltration, and to explore the underlying mechanisms.

Conclusion
In conclusion, our study found that LRRFIP1 was highly expressed in pancreatic cancer, that high LRRFIP1 expression was associated with poor overall survival and that LRRFIP1 was correlated with immune cell infiltration in pancreatic cancer.LRRFIP1 is essential for pancreatic cancer cell motility and has a minor impact on cell proliferation, which may be mediated by AKT/GSK-3β/β-catenin signaling.Our study provides evidence that LRRFIP1 may play a pivotal role in the progression of pancreatic cancer, and it may be a potential biomarker and therapeutic target for pancreatic cancer.

Fig. 1
Fig. 1 LRRFIP1 is highly expressed in pancreatic cancer tissues.A, B The mRNA expression level of LRRFIP1 in pancreatic cancer tissues and normal tissues was investigated in TCGA cohort (A) and GEO pancreatic cancer dataset GSE16515 (B).C The protein expression level of LRRFIP1 in pancreatic cancer tissues and normal tissues was analyzed in the CPTAC database.D Immunohistochemistry (IHC) analysis of LRRFIP1 in pancreatic cancer tissues and normal tissues (left), and the IHC staining was quantified (right).E ROC curve was applied to assess the accuracy of LRRFIP1 for discriminating TCGA pancreatic cancer tissues from normal tissues.* p < 0.05, *** p < 0.001 by Wilcoxon rank sum test (A, B) or Student's t-test (C, D)

Fig. 2
Fig. 2 Correlation between LRRFIP1 and prognosis in pancreatic cancer patients.A High LRRFIP1 expression indicates shorter overall survival in all pancreatic cancer patients.B-I Association of LRRFIP1 and overall survival in pancreatic cancer patients in several subgroups, including T stage: T1&T2 (B), T3&T4 (C); histologic grade: G1&G2 (D), G3&G4 (E); pathologic stage: Stage I&Stage II (F); anatomic neoplasm subdivision: head of pancreas (G); history of diabetes (H) and alcohol history (I)

Fig. 3
Fig. 3 Function and pathway enrichment analysis of LRRFIP1 in pancreatic cancer.A Significant GO terms (including BP, MF, CC, and KEGG) of the top 210 genes most positively associated with LRRFIP1.B Significant KEGG pathway of the top 210 genes most positively associated with LRRFIP1

Table 1
Univariate and multivariate analysis of the prognostic value of clinicopathological characteristics in pancreatic cancer patients' overall survival HR hazard ratio; CI confidence interval; CR complete response; PD progressive disease; SD stable disease; PR partial response